Gene expression is differently affected by pimecrolimus and betamethasone in lesional skin of atopic dermatitis.

BACKGROUND: Topical corticosteroids and calcineurin inhibitors are well-known treatments of atopic dermatitis (AD) but differ in their efficacy and side effects. We recently showed that betamethasone valerate (BM) although clinically more efficient impaired skin barrier repair in contrast to pimecrolimus in AD. OBJECTIVE: This study elucidates the mode of action of topical BM and pimecrolimus cream in AD. METHODS: Lesional AD skin samples after topical treatment with either BM or pimecrolimus were subjected to gene expression profile analysis. RESULTS: Betamethasone valerate resulted in a significant reduction in mRNA levels of genes encoding markers of immune cells and inflammation, dendritic cells, T cells, cytokines, chemokines, and serine proteases, whereas pimecrolimus exerted minor effects only. This corroborates the clinical finding that BM reduces inflammation more effectively than pimecrolimus. Genes encoding molecules important for skin barrier function were differently affected. Both BM and pimecrolimus normalized the expression of filaggrin and loricrin. BM, but not pimecrolimus, significantly reduced the expression of rate-limiting enzymes for lipid synthesis and the expression of involucrin and small proline-rich proteins, which covalently bind ceramides. This may explain the lack of restoration of functional stratum corneum layers observed after BM treatment. CONCLUSION: The gene expression profiles are consistent with our previous findings that corticosteroids may exert a more potent anti-inflammatory effect but may impair the restoration of the skin barrier. Corticosteroids are still the main treatment for severe and acutely exacerbated AD; pimecrolimus may be preferable for long-term treatment and stabilization.

Population model of the pharmacokinetics and pharmacodynamics of rivaroxaban--an oral, direct factor xa inhibitor--in healthy subjects.

OBJECTIVE: Rivaroxaban (BAY 59-7939) is an oral, direct Factor Xa (FXa) inhibitor being developed for the prevention and treatment of thromboembolic disorders. This analysis aimed to define population models for the pharmacokinetics (PK) and pharmacodynamics (PD) ofrivaroxaban in healthy males. METHODS: Non-linear, mixed-effect modeling was used to analyze rivaroxaban plasma concentration and PD data (FXa activity and clotting tests) from subjects in a phase I, multiple-ascending-dose study. Subjects received 5 mg rivaroxaban once, twice or three times daily, or 10, 20 or 30 mg rivaroxaban twice daily. RESULTS: The population PK of rivaroxaban were well described by an oral, two-compartment model with first-order absorption and elimination from the central compartment. Population mean estimates for apparent oral clearance and volume of distribution for the central compartment were 9.2 1/h and 55 1, respectively, with moderate inter-individual variability (17.4% and 30.7%, respectively). Total volume of distribution for rivaroxaban at steady state was approximately 70 1. Residual (unexplained) variability was 25%. FXa activity correlated with rivaroxaban plasma concentrations following an inhibitory Emax model; prothrombin time (PT) and rivaroxaban plasma concentrations correlated with a linear model, with a slope of 4.6 s/(100 microg/1). Inter-individual variability was low for the correlation with PT. The models derived were used to define sampling windows for population PK/PD modeling in Phase II studies. CONCLUSIONS: This analysis confirms that rivaroxaban has predictable, dose-proportional PK and PD. The linear correlation between rivaroxaban plasma concentrations and PT suggests that this test might be useful to assess rivaroxaban exposure in patients, if required.

Kinetics of standardized large volume leukapheresis (LVL) in patients do not show a recruitment phenomenon of peripheral blood progenitor cells (PBPC).

Although several studies have demonstrated the efficacy of large volume leukapheresis (LVL) to yield high numbers of peripheral blood progenitor cells (PBPC), the mechanisms of stem cell release into circulation and the postulated phenomenon of PBPC recruitment during apheresis have not been investigated in detail. Therefore, we analyzed the kinetics of stem cell enrichment in a total of 34 standardized LVL for patients with hematologic malignancies (lymphoma, multiple myeloma) and solid tumors (breast cancer, rhabdomyosarcoma). LVL was started 2 h after administration of G-CSF processing six times the patient's blood volume. Cells were sequentially collected into six bags and the numbers of leukocytes, mononuclear cells (MNC), CD34+ cells and colony-forming cells (CFU-GM) in each collection bag were analyzed. The numbers of PBPC collected demonstrated a continuous decrease starting after an early maximum during the second processed blood volume (P = 0.001). Interestingly, these kinetics of decreasing stem cell yields during LVL were similar for both entities of patients with hematologic malignancies as well as for both groups of patients with solid tumors. In summary, a recruitment phenomenon, defined as a time-dependent and LVL-induced increase of PBPC, could not be demonstrated in any of the diseases investigated.

Interleukin 10 inhibits cytokine production of human AML cells.

BACKGROUND: Unlike normal hematopoietic cells leukemic blasts from patients with AML constitutively express cytokines like IL1, GM-CSF and TNF alpha and it has been suggested that these cytokines may regulate growth and differentiation of the malignant cells. IL10 inhibits cytokine production of activated macrophages and T-helper 1 cells. We analysed whether IL10 can also suppress cytokine production and may inhibit growth of AML cells. MATERIALS AND METHODS: AML blasts of 18 patients were purified by immunomagnetic separation and cultured in serum-free medium in the presence of cytokines. The production of cytokines was analysed by ELISA, DNA synthesis by 3H-thymidine incorporation and mRNA expression of cytokine genes by semiquantitative RT-PCR. RESULTS: Our results confirm previous results that AML blasts produce a variety of cytokines such as GM-CSF, IL1 alpha, IL1 beta, IL6 and TNF alpha. AML cells were induced to proliferation by G-CSF, GM-CSF, IL3, IL1 beta and SCF to a different extent. In contrast, IL10 significantly inhibited the cytokine production at the mRNA and protein level and spontaneous thymidine uptake in a dose-dependent way. This inhibition could be abrogated by IL10 specific antibodies. CONCLUSION: These observations suggest an inhibitory effect of IL10 on the proliferation of cultured AML blasts most likely through suppression of endogenous cytokines.

Recombinant human GM-CSF following chemotherapy in high-risk AML.

Chemotherapy (CT) induced critical neutropenia remains a major dose limiting problem in acute leukemias. In order to reduce the phase of risk we gave recombinant human GM-CSF to 30 patients at high risk of early death with acute myeloid leukemia (AML). 19 patients with untreated AML and 1 patient with AML late relapse were 65+ years of age and were treated for CT by the TAD9 regimen. 10 patients at all ages had AML early or second relapse and received S-HAM CT. Starting on day 4 after CT GM-CSF 250 micrograms/m2/d was given by continuous i.v. infusion until neutrophils recovered. GM-CSF reduced the median recovery time of neutrophils by 4 days in the TAD9 and 9 days in the S-HAM CT group when compared to controls. After the CT induced aplasia 3 patients with AML showed a regrowth of their blasts which after the stop of GM-CSF was reversible in 1 patient and unaffectedly continued in 2 patients. 57% of patients attained a complete remission, and the median age of the responders was 65 (34-84) years. Remission duration was not found to be reduced. Thus, GM-CSF reduces CT toxicity with a low risk of promoting the disease and may allow more effective antileukemic treatment.

Recombinant human granulocyte-macrophage colony-stimulating factor after chemotherapy for acute leukemias at higher age or after relapse.

Chemotherapy (CT) induced critical cytopenia remains as the major dose limiting problem in the treatment of acute leukemias. This is especially true in patients at high risk of early death due to high intensity chemotherapy for relapse or to higher age. In an attempt to reduce the phase of risk we administered human recombinant GM-CSF to 23 patients at a median age of 65 (range 17-84) years including 8 acute myelogenous (AML) and 5 lymphoblastic (ALL) leukemias after early or second relapses and 10 patients of 65 years and older in primary induction treatment for AML. 3 patients with AML had prior bone marrow transplantation. 4 AML's were secondary to tumor chemotherapy (2) or myelodysplastic syndrome (2). The study was part of chemotherapeutic phase II and III studies using TAD9 for primary induction chemotherapy and S-HAM for relapses. Starting on day 4 after the end of intensive CT GM-CSF 250 micrograms/m2/day was given by continuous i.v. infusion and after recovery of neutrophils was deescalated in two 4 day steps and discontinued. 12/23 patients achieved a complete remission (CR) and median age of responders is 61 (range 17-84) years. In the group of the non-transplanted patients the median recovery time of neutrophils is reduced by one week when compared to controls receiving the same chemotherapy (p = 0.002). We observed a leukemic regrowth in 3 patients of 61, 70, and 78 years, 2 of whom having secondary AML. After discontinuation of GM-CSF the regrowth of blasts was reversible in 1 patient and continued unaffectedly in its kinetics in 2 patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Variability in 4-hydroperoxycyclophosphamide activity during clinical purging for autologous bone marrow transplantation.

We examined the effects of varying incubation conditions on the in vitro activity of 4-hydroperoxycyclophosphamide (4HC). 4HC activity against CFU-GM and against the K562 tumor cell line decreased with increasing the RBC concentration of the incubation mixture. Increasing the concentration of nucleated bone marrow cells in the incubation mixture also decreased the 4HC activity. Evaluation of 53 consecutive patients undergoing autologous bone marrow transplantation (BMT) revealed that the incubation RBC concentration during clinical purging showed a similar effect on CFU-GM recovery. Aldehyde dehydrogenase content of RBCs and nucleated marrow cells appears to be the cause of the inhibition of 4HC activity. Although there was no difference in individual CFU-GM sensitivity to 4HC among normals, previously treated patients undergoing autologous BMT showed significant variability in CFU-GM sensitivity to 4HC. The combined effects of incubation RBC concentration and individual patient 4HC sensitivity appear to account for most of the variability in CFU-GM recovery and speed of hematologic recovery after clinical purging with 4HC.

CFU-GM content of bone marrow graft correlates with time to hematologic reconstitution following autologous bone marrow transplantation with 4-hydroperoxycyclophosphamide-purged bone marrow.

Autologous bone marrow transplants (BMTs) can repopulate the hematologic system of patients treated with marrow-ablative chemotherapy and/or radiotherapy. However, treatment of the bone marrow graft to eliminate residual tumor cells prior to reinfusion can delay the return of peripheral blood elements, presumably from damage to or loss of hematopoietic stem cells responsible for hematologic recovery. To develop a model predictive of hematologic recovery, we studied the progenitor cell contents of 4-hydroperoxycyclophosphamide (100 micrograms/mL)-purged bone marrow grafts of 40 consecutive patients undergoing autologous BMT at this center. Granulocyte-macrophage colonies (CFU-GM) were grown from all grafts after treatment with this chemotherapeutic agent, but erythroid (BFU-E) and mixed (CFU-GEMM) colonies were grown from only 44% and 33% of the grafts respectively. The recovery of CFU-GM after purging ranged from 0.07% to 23%. The logarithm of CFU-GM content of the treated grafts was linearly correlated with the time to recovery of peripheral blood leukocytes (r = -0.80), neutrophils (r = -0.79), reticulocytes (r = -0.60), and platelets (r = -0.66). The CFU-GM content of purged autologous bone marrow grafts may reflect the hematopoietic stem cell content of the grafts and thus predict the rate of hematologic recovery in patients undergoing autologous BMT.